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Acceptor Substrate Discrimination in Phosphatidyl-myo-inositol Mannoside Synthesis

STRUCTURAL AND MUTATIONAL ANALYSIS OF MANNOSYLTRANSFERASE CORYNEBACTERIUM GLUTAMICUM PimB′
  • Sarah M. Batt
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Talat Jabeen
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Arun K. Mishra
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Natacha Veerapen
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Karin Krumbach
    Affiliations
    Institut für Biotechnologie I, Forschungszentrum Jülich, D-52425 Jülich, Germany
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  • Lothar Eggeling
    Affiliations
    Institut für Biotechnologie I, Forschungszentrum Jülich, D-52425 Jülich, Germany
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  • Gurdyal S. Besra
    Correspondence
    Recipient of support in the form of a Personal Research Chair from James Bardrick, Royal Society Wolfson Research Merit Award, as a former Lister Institute-Jenner Research Fellow. To whom correspondence may be addressed: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Tel.: 44-121-4145895; Fax: 44-121-4145925;
    Footnotes
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Klaus Fütterer
    Correspondence
    To whom correspondence may be addressed: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Tel.: 44-121-4145895; Fax: 44-121-4145925;
    Footnotes
    Affiliations
    From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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  • Author Footnotes
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1S–5S.
    1 Supported by Wellcome Trust Grant 081569/Z/06/Z, and by Advantage West Midlands through the Birmingham Science City Translational Medicine Platform.
Open AccessPublished:September 15, 2010DOI:https://doi.org/10.1074/jbc.M110.165407
      Long term survival of the pathogen Mycobacterium tuberculosis in humans is linked to the immunomodulatory potential of its complex cell wall glycolipids, which include the phosphatidylinositol mannoside (PIM) series as well as the related lipomannan and lipoarabinomannan glycoconjugates. PIM biosynthesis is initiated by a set of cytosolic α-mannosyltransferases, catalyzing glycosyl transfer from the activated saccharide donor GDP-α-d-mannopyranose to the acceptor phosphatidyl-myo-inositol (PI) in an ordered and regio-specific fashion. Herein, we report the crystal structure of mannosyltransferase Corynebacterium glutamicum PimB′ in complex with nucleotide to a resolution of 2.0 Å. PimB′ attaches mannosyl selectively to the 6-OH of the inositol moiety of PI. Two crystal forms and GDP- versus GDP-α-d-mannopyranose-bound complexes reveal flexibility of the nucleotide conformation as well as of the structural framework of the active site. Structural comparison, docking of the saccharide acceptor, and site-directed mutagenesis pin regio-selectivity to a conserved Asp residue in the N-terminal domain that forces presentation of the correct inositol hydroxyl to the saccharide donor.

      Introduction

      The cell envelope of Mycobacterium tuberculosis, the infectious agent causing tuberculosis, contains a variety of glycolipids that play a central role in subverting the host's immune response and thus help establish a long lasting latent infection, a hallmark of the pathophysiology of tuberculosis (
      • Józefowski S.
      • Sobota A.
      • Kwiatkowska K.
      ,
      • Guenin-Macé L.
      • Siméone R.
      • Demangel C.
      ). Phosphatidylinositol mannosides (PIMs)
      The abbreviations used are: PIM
      phosphatidylinositol mannoside
      PI
      phosphatidyl-myo-inositol-1-phosphate
      GDP-Man
      guanosine-5′-diphosphate-α-d-mannose
      LM
      lipomannan
      LAM
      lipoarabinomannan
      GT4
      glycosyltransferase family 4
      NCS
      non-crystallographic symmetry
      r.m.s.
      root mean square
      SeMet
      selenomethionine
      H-bond
      hydrogen bond
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      represent a series of glycolipids that comprise a phosphatidyl-myo-inositol (PI) core, an acylated mannosyl group attached to the 2-hydroxyl of inositol, and a mannosyl-oligosaccharide of variable length attached to the inositol 6-hydroxyl (Fig. 1) (
      • Berg S.
      • Kaur D.
      • Jackson M.
      • Brennan P.J.
      ). A precursor of the more complex lipomannan (LM) and lipoarabinomannan (LAM) glycolipids, PIMs have been shown to influence both innate (
      • Doz E.
      • Rose S.
      • Court N.
      • Front S.
      • Vasseur V.
      • Charron S.
      • Gilleron M.
      • Puzo G.
      • Fremaux I.
      • Delneste Y.
      • Erard F.
      • Ryffel B.
      • Martin O.R.
      • Quesniaux V.F.
      ,
      • Rhoades E.R.
      • Archambault A.S.
      • Greendyke R.
      • Hsu F.F.
      • Streeter C.
      • Byrd T.F.
      ) and adaptive immunity of the host (
      • Fischer K.
      • Scotet E.
      • Niemeyer M.
      • Koebernick H.
      • Zerrahn J.
      • Maillet S.
      • Hurwitz R.
      • Kursar M.
      • Bonneville M.
      • Kaufmann S.H.
      • Schaible U.E.
      ,
      • de la Salle H.
      • Mariotti S.
      • Angenieux C.
      • Gilleron M.
      • Garcia-Alles L.F.
      • Malm D.
      • Berg T.
      • Paoletti S.
      • Maître B.
      • Mourey L.
      • Salamero J.
      • Cazenave J.P.
      • Hanau D.
      • Mori L.
      • Puzo G.
      • De Libero G.
      ,
      • Driessen N.N.
      • Ummels R.
      • Maaskant J.J.
      • Gurcha S.S.
      • Besra G.S.
      • Ainge G.D.
      • Larsen D.S.
      • Painter G.F.
      • Vandenbroucke-Grauls C.M.
      • Geurtsen J.
      • Appelmelk B.J.
      ,
      • Mahon R.N.
      • Rojas R.E.
      • Fulton S.A.
      • Franko J.L.
      • Harding C.V.
      • Boom W.H.
      ).
      Figure thumbnail gr1
      FIGURE 1Schematic diagram of PIMs and pathway of PIM synthesis. ManT and AraT, generic mannosyl- and arabinosyltransferase enzyme, respectively. LM, lipomannan; LAM, lipoarabinomannan.
      PIM biosynthesis begins by consecutive transfer of two mannosyl units from activated sugar-nucleotide (GDP-Man) to PI, catalyzed by cytoplasmic α-mannosyltransferases (Fig. 1). Attachment of the first mannosyl residue to the 2-hydroxyl of the inositol ring, resulting in PIM1, is catalyzed by PimA (M. tuberculosis Rv2610c, Mycobacterium smegmatis MSMEG_2935) (
      • Guerin M.E.
      • Schaeffer F.
      • Chaffotte A.
      • Gest P.
      • Giganti D.
      • Korduláková J.
      • van der Woerd M.
      • Jackson M.
      • Alzari P.M.
      ,
      • Korduláková J.
      • Gilleron M.
      • Mikusova K.
      • Puzo G.
      • Brennan P.J.
      • Gicquel B.
      • Jackson M.
      ), followed by acylation of the 2-mannose (by M. tuberculosis Rv2611c) (
      • Korduláková J.
      • Gilleron M.
      • Puzo G.
      • Brennan P.J.
      • Gicquel B.
      • Mikusová K.
      • Jackson M.
      ) to yield monoacylated PIM1 (Ac1PIM1). The second mannosyl residue is attached at the 6-hydroxyl, to yield Ac1PIM2, a reaction catalyzed by PimB′ (M. tuberculosis Rv2188c, M. smegmatis MSMEG_4253, Corynebacterium glutamicum NCgl2106) (
      • Lea-Smith D.J.
      • Martin K.L.
      • Pyke J.S.
      • Tull D.
      • McConville M.J.
      • Coppel R.L.
      • Crellin P.K.
      ,
      • Guerin M.E.
      • Kaur D.
      • Somashekar B.S.
      • Gibbs S.
      • Gest P.
      • Chatterjee D.
      • Brennan P.J.
      • Jackson M.
      ,
      • Mishra A.K.
      • Batt S.
      • Krumbach K.
      • Eggeling L.
      • Besra G.S.
      ). The designation PimB had originally been assigned to open reading frame Rv0557, also encoding an α-mannosyltransferase. Subsequently, Rv0557 was found to be dispensable for synthesis of Ac1PIM2, and the corresponding protein has since been renamed MgtA (
      • Mishra A.K.
      • Batt S.
      • Krumbach K.
      • Eggeling L.
      • Besra G.S.
      ,
      • Schaeffer M.L.
      • Khoo K.H.
      • Besra G.S.
      • Chatterjee D.
      • Brennan P.J.
      • Belisle J.T.
      • Inamine J.M.
      ,
      • Tatituri R.V.
      • Illarionov P.A.
      • Dover L.G.
      • Nigou J.
      • Gilleron M.
      • Hitchen P.
      • Krumbach K.
      • Morris H.R.
      • Spencer N.
      • Dell A.
      • Eggeling L.
      • Besra G.S.
      ). For consistency with the recent literature, we retain the designation PimB′ for Rv2188c (and its orthologs in M. smegmatis and C. glutamicum) (
      • Guerin M.E.
      • Kaur D.
      • Somashekar B.S.
      • Gibbs S.
      • Gest P.
      • Chatterjee D.
      • Brennan P.J.
      • Jackson M.
      ,
      • Mishra A.K.
      • Batt S.
      • Krumbach K.
      • Eggeling L.
      • Besra G.S.
      ,
      • Mishra A.K.
      • Klein C.
      • Gurcha S.S.
      • Alderwick L.J.
      • Babu P.
      • Hitchen P.G.
      • Morris H.R.
      • Dell A.
      • Besra G.S.
      • Eggeling L.
      ).
      Prolonged incubation of Ac1PIM2 with PimB′ or PimA does not lead to further extension of Ac1PIM2 (
      • Guerin M.E.
      • Kaur D.
      • Somashekar B.S.
      • Gibbs S.
      • Gest P.
      • Chatterjee D.
      • Brennan P.J.
      • Jackson M.
      ). Instead, extending the mannosyl chain at the 6-OH requires a distinct set of mannosyltransferases. For instance, bioinformatic analysis of the genome of M. tuberculosis CDC1551 led to the identification of PimC, which catalyzes synthesis of the trimannoside Ac3PIM3 (
      • Kremer L.
      • Gurcha S.S.
      • Bifani P.
      • Hitchen P.G.
      • Baulard A.
      • Morris H.R.
      • Dell A.
      • Brennan P.J.
      • Besra G.S.
      ). Still, the pimC deletion in Mycobacterium bovis bacille Calmette-Guérin does not interrupt formation of higher PIMs, LM or LAM, for which PIMn (where n = 1–3) is considered a precursor, whereas genes orthologous to pimC were found in only 22% of clinical isolates. Thus, compensatory synthetic pathways must exist. Higher order PIM, LM, and LAM depend on glycosyl transfer from the lipid-saccharide donor C30/C50-P-Man and membrane-embedded glycosyltransferases, including PimE (
      • Morita Y.S.
      • Sena C.B.
      • Waller R.F.
      • Kurokawa K.
      • Sernee M.F.
      • Nakatani F.
      • Haites R.E.
      • Billman-Jacobe H.
      • McConville M.J.
      • Maeda Y.
      • Kinoshita T.
      ), with evidence for pathway bifurcation at Ac2PIM4 (
      • Morita Y.S.
      • Patterson J.H.
      • Billman-Jacobe H.
      • McConville M.J.
      ,
      • Patterson J.H.
      • Waller R.F.
      • Jeevarajah D.
      • Billman-Jacobe H.
      • McConville M.J.
      ).
      The three-dimensional structures of soluble glycosyltransferases display only two fundamental fold topologies, termed GT-A and GT-B (
      • Liu J.
      • Mushegian A.
      ), contrasting with the diversity of protein folds among glycoside hydrolase enzymes (
      • Lairson L.L.
      • Henrissat B.
      • Davies G.J.
      • Withers S.G.
      ). According to the sequence-based classification of carbohydrate-active enzymes, PimB′ (Rv2188c) belongs to glycosyltransferase family 4 (GT4; see the CAZy database) (
      • Coutinho P.M.
      • Deleury E.
      • Davies G.J.
      • Henrissat B.
      )). Encompassing a diverse range of enzymatic activities and substrate specificities, GT4 family enzymes assume the GT-B fold, with known structures for about half a dozen family members (
      • Martinez-Fleites C.
      • Proctor M.
      • Roberts S.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      ,
      • Guerin M.E.
      • Kordulakova J.
      • Schaeffer F.
      • Svetlikova Z.
      • Buschiazzo A.
      • Giganti D.
      • Gicquel B.
      • Mikusova K.
      • Jackson M.
      • Alzari P.M.
      ,
      • Ruane K.M.
      • Davies G.J.
      • Martinez-Fleites C.
      ,
      • Vetting M.W.
      • Frantom P.A.
      • Blanchard J.S.
      ,
      • Chua T.K.
      • Bujnicki J.M.
      • Tan T.C.
      • Huynh F.
      • Patel B.K.
      • Sivaraman J.
      ). The GT-B topology is characterized by two Rossmann fold-like domains, where donor and acceptor substrates bind in the central cleft between the two domains. In GT4 family transferases, the C-terminal domain provides the majority of contacts for the nucleoside-diphosphate-saccharide donor substrate, whereas the diverse acceptor-substrates bind to the more variable N-terminal domain. A hinge region allows for conformational flexibility between the two domains, which can be dramatic, as is illustrated by the structures of ligand-free and substrate-bound C. glutamicum MshA (Protein Data Bank entries 3c48 and 3c4v (
      • Vetting M.W.
      • Frantom P.A.
      • Blanchard J.S.
      )). Between these two states, the relative orientation of the domains changes by a 97° rotational movement. Similar if less dramatic examples of interdomain flexibility have been observed in structures of several other GT4 family members.
      Recently determined structures of GT4 family enzymes include that of M. smegmatis PimA, the enzyme catalyzing transfer of the first mannosyl group to PI (
      • Guerin M.E.
      • Kordulakova J.
      • Schaeffer F.
      • Svetlikova Z.
      • Buschiazzo A.
      • Giganti D.
      • Gicquel B.
      • Mikusova K.
      • Jackson M.
      • Alzari P.M.
      ). This structure shed light on the mode of nucleotide binding and suggested a model for recognition of the acceptor substrate. Although PimA and PimB′ utilize the same donor substrate, their acceptor specificity is distinct. PimB′ appears unable to mannosylate phosphatidyl-myo-inositol (or myo-inositol-1-phosphate) on the 2-hydroxyl, whereas PimA does not mannosylate PI or PIM1 on the 6-hydroxyl.
      In order to clarify structural differences between PimA and PimB′ that could explain regio-selectivity, we undertook the structure determination of PimB′. Attempts to generate recombinant protein of M. tuberculosis PimB′ (Rv2188c) were unsuccessful, but overexpression of the C. glutamicum ortholog (NCgl2106) in Escherichia coli yielded soluble protein that crystallized when incubated with the donor substrate. Herein, we report the crystal structure of C. glutamicum PimB′ in complex with nucleotide to a resolution of 2.0 Å, revealing flexibility of both the nucleotide conformation and the structural framework of the active site. The results of our site-directed mutational analysis are consistent with a substrate-mediated SNi (internal return) reaction mechanism and indicate that a conserved aspartic acid in the acceptor-binding domain is critical in determining regio-selectivity of mannosyl transfer.

      DISCUSSION

      PIM biosynthesis occurs through a series of enzyme-catalyzed reactions during which PI is decorated on the inositol moiety with mannosyl groups in an ordered and regio-selective manner. The present structure in combination with the mutational analysis sheds light on how regio-selectivity is achieved and provides indirect evidence for the hypothesis that catalysis occurs through a substrate-mediated SNi internal return mechanism.
      In terms of fold and overall structure, PimB′ conforms to the paradigm of GT-B glycosyltransferases. However, a couple of observations are noteworthy. First, nucleotide binding did not result in crystallization of a closed configuration of the enzyme, in contrast to the closed states observed for the nucleotide-bound complexes of PimA, MshA, OtsA, or WaaG (
      • Martinez-Fleites C.
      • Proctor M.
      • Roberts S.
      • Bolam D.N.
      • Gilbert H.J.
      • Davies G.J.
      ,
      • Guerin M.E.
      • Kordulakova J.
      • Schaeffer F.
      • Svetlikova Z.
      • Buschiazzo A.
      • Giganti D.
      • Gicquel B.
      • Mikusova K.
      • Jackson M.
      • Alzari P.M.
      ,
      • Vetting M.W.
      • Frantom P.A.
      • Blanchard J.S.
      ,
      • Gibson R.P.
      • Turkenburg J.P.
      • Charnock S.J.
      • Lloyd R.
      • Davies G.J.
      ). However, it seems improbable that PimB′ is unable to close the active site cleft, given the close similarity to the structural homologs. Small changes in orientation of the domains between the orthorhombic and triclinic crystal form provide direct evidence for conformational flexibility. More importantly, the G20W mutation leads to a 97% drop in activity but binds the donor with an affinity similar to that of wild type PimB′ (Table 2). The fact that a bulky side chain in the center of the cleft is tolerated in terms of nucleotide binding but abrogates activity is consistent with the notion that it impedes the closing motion.
      Second, the conformational flexibility observed in the β5-α5 loop of the active site cleft has not been observed previously in GT4 glycosyltransferases. The shift of individual side chains and the corresponding change of intramolecular contacts seem significant. For instance, the position of the Trp124 Cα is shifted by 7 Å, and its side chain makes contacts with different parts of the structure in the two crystal forms. We probed whether helix α5′ is required for activity by substituting glycine at position 123 with proline, but the resulting drop of activity is only about 15% (Table 2). Furthermore, His120 at the N terminus of helix α5′ is only ordered in the triclinic structure and disordered when helix α5′ forms. Substituting His120 by serine reduced activity by about 20% (Table 2), whereas the CD spectrum of the mutant G123P is virtually identical to that of wild-type PimB′ (supplemental Fig. 4S). In light of these data, we hypothesize that the conformational flexibility of the β5-α5 loop works in concert with the opening/closing motion of the active site cleft upon substrate binding but influences catalysis only in an indirect fashion if at all.
      PimB′ is a GT-B glycosyltransferase that retains the stereochemical configuration of the anomeric carbon of the donor saccharide. The catalytic reaction mechanism of retaining GT-B transferases has remained something of a mystery. In glycoside hydrolases, retention of the anomeric configuration occurs through an SN2-like double displacement mechanism, mediated by a pair of side chain carboxylates and involving a covalent sugar-enzyme intermediate (
      • Zechel D.L.
      • Withers S.G.
      ). However, none of the inhibitor- or substrate mimic-bound complexes solved to date has provided structural evidence to support such a scenario in GT-B glycosyltransferases (
      • Lairson L.L.
      • Henrissat B.
      • Davies G.J.
      • Withers S.G.
      ,
      • Sheng F.
      • Jia X.
      • Yep A.
      • Preiss J.
      • Geiger J.H.
      ,
      • Steiner K.
      • Hagelueken G.
      • Messner P.
      • Schäffer C.
      • Naismith J.H.
      ,
      • Frantom P.A.
      • Coward J.K.
      • Blanchard J.S.
      ). Instead, a substrate-mediated SNi (internal return) mechanism has been invoked (reviewed in Ref.
      • Lairson L.L.
      • Henrissat B.
      • Davies G.J.
      • Withers S.G.
      ). This mechanism involves three tightly linked reaction steps (Scheme 1): first, decay of the sugar-phosphate bond, leaving a (solvent-separated) ion pair of the oxocarbenium ion-like saccharide and the negatively charged phosphate (panels 2 and 3); second, deprotonation of the acceptor hydroxyl by the phosphate leaving group; third, nucleophilic attack by the acceptor on the oxocarbenium ion (panels 3 and 4). The first step is rate-limiting, whereas the subsequent steps must occur on a time scale shorter than solvent attack or ion pair reorganization (
      • Lairson L.L.
      • Henrissat B.
      • Davies G.J.
      • Withers S.G.
      ). If correct, this mechanism imposes constraints on the geometry of donor and acceptor substrate in the ternary complex, in that the leaving group must be sufficiently close to the acceptor to be able to deprotonate the attacking acceptor hydroxyl.
      Figure thumbnail grs1
      SCHEME 1Schematic representation of steps of the SNi-like (internal return) reaction mechanism leading to retention of the α-configuration of the anomeric carbon of the donor saccharide. This scheme was adapted from Ref.
      • Errey J.C.
      • Lee S.S.
      • Gibson R.P.
      • Martinez Fleites C.
      • Barry C.S.
      • Jung P.M.
      • O'Sullivan A.C.
      • Davis B.G.
      • Davies G.J.
      .
      In our model of the acceptor-bound complex, the 6-OH of inositol (Oi6) is positioned within ∼4 Å of the C1 carbon of the donor mannose and within 5 Å of the β-phosphate. The distance vectors Oi6-C1 and C1-β-phosphate enclose an angle of ∼105°. Thus, the geometric configurations of the substrates in the ternary complex model are compatible with the requirements of the SNi reaction mechanism. Our mutagenesis data provide further supporting evidence for a substrate-dependent reaction mechanism. For instance, the most obvious candidate to act as a catalytic nucleophile in an SN2-like mechanism is Glu290, a carboxylate that is strictly conserved across GT4 family enzymes and positioned close (∼4.5 Å) to the sugar-phosphate bond. Switching this residue to glutamine, which should completely impair nucleophilic attack, decreases activity by about 95% while leaving substrate binding largely intact (<3-fold increase of Kd). Importantly, the residual activity is about 10 times above base-line level and can be reduced further by the E290D substitution (Table 2). In contrast, the substitution of highly conserved His118 by serine decreases activity essentially to base-line level. In the “open” configuration, His118 does not contact the donor substrate, but in the “closed” model, it contacts the C6 hydroxyl of the nucleotide-linked mannose. His118 is positioned closely opposite to Ile21 (closest contact 3.9 Å). Substitution of Ile21 with either Ser or Ala diminishes activity to about 2.5%. The close proximity of a hydrophobic side chain to His118 suggests that the latter is pushed to its non-ionized state, which could enable the His side chain to act as a nucleophile. However, in our modeled ternary complex as in the experimental structures of OtsA (Protein Data Bank entry 2wtx) or MshA (Protein Data Bank entry 3c4v), the position of His118 relative to the nucleotide makes such a role improbable.
      In their structural evaluation of PimA, Guerin et al. (
      • Guerin M.E.
      • Kordulakova J.
      • Schaeffer F.
      • Svetlikova Z.
      • Buschiazzo A.
      • Giganti D.
      • Gicquel B.
      • Mikusova K.
      • Jackson M.
      • Alzari P.M.
      ) identified Tyr9 as a residue that might form a stacking interaction with the inositol ring of PI and thus may be key to acceptor specificity. This tyrosine is located in the β1-α1 loop and corresponds to Asp13 in PimB′. Within the sequences of PimB′ and PimA enzymes, respectively, the Asp and Tyr are strictly conserved at this position (supplemental Fig. 1S). In the model of the ternary complex, Asp13 is positioned within H-bond distance to the 3- and 4-OH of the inositol moiety (i.e. the only inositol hydroxyls that are in an equatorial configuration). Such a bidentate interaction would ensure that inositol presents the axial 6-OH rather than the 2-OH to the C1 carbon of mannose. Indeed, swapping the positions of 3-OH and 4-OH of inositol would present the 5-OH to C1 of mannose rather than 2-OH. Probing Asp13 by mutagenesis, it is intriguing that even the most conservative substitution, aspartate to asparagine, reduces activity by 98.5% relative to wild type. Indeed, this subtle change knocks down activity as effectively as the more drastic Asp to Ala substitution (Table 2). Residues in the immediate vicinity (Asn12, Ile21, and Gln22) are similarly sensitive to substitutions, causing a reduction of activity by at least 70%. We also probed whether the specificity of PimB′ can be switched to mannosylation of the 2-OH by substituting Asp13 by tyrosine. However, the result was a knockdown of activity to blank level (Table 2). Thus, residues in the β1-α1 loop play a key role in acceptor recognition, but the single substitution on position 13 is not sufficient to switch acceptor specificity.
      The deleterious effect of the D13N mutation stresses the significance of Asp13 in determining acceptor specificity, although the mechanistic implication of this substitution is not clear because an asparagine should still be able to form H-bonds with the inositol hydroxyl groups. However, the effect is reminiscent of an analogous mutation in glycosidase family GH68. There, substitution of a strictly conserved Asp by Asn, which formed strong H-bonds with saccharide substrate hydroxyls but, for geometric reasons, could not be a nucleophile or general acid/base, reduced activity by 2 orders of magnitude in terms of kcat. This effect was attributed to stabilization of the oxocarbenium ion transition state by the negatively charged carboxylate (
      • Meng G.
      • Fütterer K.
      ,
      • Yanase H.
      • Maeda M.
      • Hagiwara E.
      • Yagi H.
      • Taniguchi K.
      • Okamoto K.
      ). Lack of sequence conservation across GT4 family enzymes appears to rule out a fundamental role of Asp13 in a general reaction mechanism of GT4 enzymes. However, we note that PimA extends a glutamic acid side chain (residue 199) toward the inositol ring when superimposed with our model of the PimB′ ternary complex. This glutamic acid, which is critical for activity (
      • Guerin M.E.
      • Schaeffer F.
      • Chaffotte A.
      • Gest P.
      • Giganti D.
      • Korduláková J.
      • van der Woerd M.
      • Jackson M.
      • Alzari P.M.
      ), is located in the β8-α8 loop and, in the structural superposition, lines up with Val208 of PimB′. Our model suggests that Glu199 could form H-bond contacts with the equatorial OH groups of inositol if the inositol moiety is oriented such that the 2-OH is presented to the C1 of the donor saccharide. Thus, the active site cleft of PimA includes a side chain that could play a role functionally analogous to that of Asp13 in PimB′.
      The dual conformation of the pyrophosphate in complexes co-crystallized with GDP-Man is intriguing. This feature could be mechanistically linked to release of the donor mannosyl group following decay of the sugar-phosphate bond and transfer to inositol. PimB′·GDP complexes have a pronounced preference for the “OtsA-like” nucleotide conformation, whereas GDP-Man bound PimB′ displayed either the superposition of the OtsA- and PimA-like states or only the PimA-like state. Density for the mannose group is completely missing in the orthorhombic complex. However, the presence of a disordered sugar moiety can be inferred on the one hand from the pronounced preference for a dual nucleotide conformation in the GDP-Man co-crystals and, second, from the positioning of an extra water molecule between β-phosphate and Glu290 in the GDP co-crystals (W4 in Fig. 4C). These observations were consistent between at least two different crystals of each complex. Between the PimA- and OtsA-like conformations, the β-phosphate shifts away from Arg206 by about 0.5–0.6 Å, weakening the corresponding ionic interaction, which would facilitate increased mobility of Arg206. Such increased mobility of this side chain could promote release of the transferred mannosyl group from the confines of the donor substrate-binding cavity.

      Conclusions

      Biosynthesis of PIM occurs through a series of glycosyl transfer reactions that initially take place at the cytoplasmic side of the membrane. The final product is presented on the extracellular face of the membrane, and hence flippase-mediated membrane translocation of either the final or an intermediate product must occur along the way (
      • Berg S.
      • Kaur D.
      • Jackson M.
      • Brennan P.J.
      ). We consider the following scenario probable. PimA, PimB′, and PimC act on acceptor substrates (PI and intermediate forms of PIM) that are anchored in the cytoplasmic leaflet of the membrane. The active site geometry of PimB′ and its electrostatic surface potential (supplemental Fig. 5S) are compatible with the notion of transient membrane association, potentially in the orientation shown in Fig. 5A. Liposome binding data recorded for PimA further support this scenario (
      • Guerin M.E.
      • Kordulakova J.
      • Schaeffer F.
      • Svetlikova Z.
      • Buschiazzo A.
      • Giganti D.
      • Gicquel B.
      • Mikusova K.
      • Jackson M.
      • Alzari P.M.
      ). PimB′ differs from PimA in several structural details that reflect their distinct acceptor substrate specificities. Chief among them is the lack of the extended β3-α3 loop, which contributes to acceptor binding in PimA but, based on geometry, cannot play such a role in PimB′. Second, the active site cleft shows a more open surface, to accommodate the mannose attached to the 2-position of inositol. Third, we show that Asp13, which is critical for activity, cannot be swapped for the corresponding tyrosine in PimA. We postulate that Asp13 is chiefly responsible for regio-specific orientation of inositol relative to the donor saccharide. Our mutagenesis data also lend indirect support to the hypothesis that glycosyl transfer with retention of the α-configuration of the anomeric carbon occurs through a substrate-mediated SNi-type mechanism.

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